IAI Accepts, published online ahead of print on 28 April 2014 Infect. Immun. doi:10.1128/IAI.01517-13 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Chemokines and antimicrobial peptides cag-dependent early response to
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Helicobacter pylori infection in primary human gastric epithelial cells
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Pascale Mustapha1, Isabelle Paris1, 2, Magali Garcia1, 2, Cong Tri Tran1, 2, Julie Cremniter1, 2,
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Martine Garnier1, 2, Jean-Pierre Faure2, Thierry Barthes3, Ivo G. Boneca4, 5, Franck Morel1, Jean-
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Claude Lecron1, 2, Christophe Burucoa1, 2, Charles Bodet1#.
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1. Laboratoire Inflammation, Tissus Epithéliaux et Cytokines (LITEC - EA 4331), Université de
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Poitiers, France.
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2. Centre Hospitalier Universitaire de Poitiers, France.
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3. Polyclinique de Poitiers, France.
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4. Institut Pasteur, Unité de Biologie et Génétique de la Paroi Bactérienne, Paris, France.
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5. INSERM, Equipe Avenir, Paris, France.
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# Address correspondence to Charles Bodet, [email protected]
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Running Head: Primary gastric cell response to H. pylori
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1
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Abstract
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Helicobacter pylori infection systematically causes chronic gastric inflammation that can persist
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asymptomatically or evolve towards more severe gastroduodenal pathologies such as ulcer,
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MALT lymphoma or gastric cancer. The cag pathogenicity island (cag-PAI) of H. pylori allows
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for translocation of the virulent protein CagA and fragments of peptidoglycan into host cells,
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thereby inducing production of chemokines, cytokines and antimicrobial peptides. In order to
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characterize inflammatory response to H. pylori, a new experimental protocol for isolating and
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culturing primary human gastric epithelial cells was established using pieces of stomach from
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patients who had undergone sleeve gastrectomy. Isolated cells expressed markers indicating that
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they were mucin-secreting epithelial cells. Challenge of primary epithelial cells with H. pylori
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B128 underscored early dose-dependent induction of mRNA expression of the inflammatory
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mediators CXCL1-3, CXCL5, CXCL8, CCL20, BD2 and TNFĮ. In AGS cells, significant
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expression of only CXCL5 and CXCL8 was observed following infection suggesting that these
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cells were less reactive than primary epithelial cells. Infection of both cellular models with H.
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pylori B128ǻcagM, a cag-PAI mutant, resulted in weak inflammatory mediator mRNA
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induction. At 24h post-infection of primary epithelial cells with H. pylori, inflammatory mediator
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production was largely due to cag-PAI-substrate-independent virulence factors. Thus, H. pylori
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cag-PAI substrate appears to be involved in eliciting an epithelial response during the early
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phases of infection. Afterwards, other virulence factors of the bacterium take over in
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development of the inflammatory response. Using a relevant cellular model, this study provides
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new information on the modulation of inflammation during H. pylori infection.
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Introduction
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It is now widely established that infection with Helicobacter pylori is the leading cause of
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chronic gastritis, duodenal ulcers and gastric cancer. This Gram-negative bacterial pathogen
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selectively colonizes the human gastric epithelium, thereby inducing an inflammatory response
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characterized by massive neutrophil infiltration and large-scale production of cytokines and
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chemokines, mainly interleukin (IL)-8 (CXCL8).
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The cag Pathogenicity Island (cag-PAI) is one of the most widely studied virulence
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factors of H. pylori. This 37 kb DNA segment encodes a type IV secretion apparatus (TIVSS)
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that delivers virulent factors such as the CagA protein as well as fragments of peptidoglycan (PG)
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into host cells (1-3). CagM is a 43.7 kDa protein, which is also encoded by cag-PAI. Along with
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CagX and CagT, CagM forms an outer membrane-associated TIVSS sub-complex (4).
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Systematic mutagenesis of cag-PAI genes showed that H. pylori ǻcagM strains cannot perform
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efficient translocation of CagA or PG into epithelial cells (3). CagA and CagM are consequently
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important when assessing the virulence of H. pylori strains.
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In order to study the interactions between cag-PAI and epithelial cells, several cell line
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models derived from gastric adenocarcinoma such as the AGS or KATO-3 cell lines have been
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used. Even though these models have clarified many aspects of the changes triggered by H. pylori
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at the cellular level such as establishment of the inflammatory response, at a later time these cell
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lines were found to have lost some of the key elements in signaling pathways involved in cellular
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responses to H. pylori infection. For example, the AGS cell line lacks the Toll-like Receptor 2
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(TLR2) and the MD2 cofactor, which is essential for lipopolysaccharide (LPS) recognition by
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TLR4 (5).
3
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As a result, so as to obtain a cellular model more relevant to define and characterize the
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pathophysiological processes associated with H. pylori infection, development of primary human
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epithelial cell models was considered necessary. Even though primary culture of human gastric
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epithelial cells has been limited, mainly on account of the unavailability of sufficient quantities of
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human gastric tissue and the difficulty of isolating and expanding these cells, a few models have
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been developed using organ donors (6), fetal tissue (7) and gastric biopsies (8). Isolation of
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gastric epithelial cells from organ donors was used to study the characteristics of attachment of
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H. pylori to cultured cells (9) and its effects on gastrin secretion (10). In another study, Basque et
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al. developed a primary epithelial cell model from legally aborted fetuses (7), and it was used to
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study the secretion of pepsinogen and gastric lipase (11) and to examine the effects of growth
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factors on cell proliferation and regeneration (12). In a third approach, Smoot et al. developed a
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protocol for primary culture of epithelial cells using gastric biopsies (8). Using the same model,
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Bäckhed et al. reported that H. pylori infection of primary cells induced a regulated production of
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IL-6, IL-8, and tumor necrosis factor–Į, whereas infection of cell lines only resulted in IL-8
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production (13). A CagA-dependent induction of cytoskeletal rearrangements and alterations in
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cell junctions was likewise reported with regard to this model (14). However, given the low yield
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in primary cells, these partial results needed to be complemented by parallel studies on cell lines.
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And even though the aforementioned models have clearly provided valuable insights into the
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functioning of normal gastric epithelial cells, up until now the inflammatory response associated
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with H. pylori has remained poorly investigated in primary cells. The aim of this study is to
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characterize H. pylori-associated inflammatory reactions by applying a new protocol allowing for
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isolation and culture of human primary gastric epithelial cells (PGEC) using normal gastric tissue
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obtained from obese subjects having undergone gastric sleeve surgery. Isolation of PGEC from 4
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large pieces of human stomach is an original approach that has contributed to the development of
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a pertinent in vitro cell model facilitating study of the expression of the antimicrobial peptides,
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chemokines and cytokines induced by H. pylori.
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Materials and Methods
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Human stomach samples. The use of stomach samples for this study was approved by the ethics
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committee of Poitiers hospital. Under fully informed consent, normal human antra were obtained
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from H. pylori-negative subjects who had undergone gastric sleeve surgery.
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Isolation and culture of PGEC from human stomach. Pieces of antrum were
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abundantly washed with phosphate buffered saline solution (PBS) (Gibco) to remove blood and
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mucous and then cut along the surgical crease. Fat and muscle layers were discarded. The mucosa
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was carefully dissected from underlying submucosa and minced into small fragments of about 2
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mm3 using scalpel blades. Tissues were digested in 0.5 mg/ml of collagenase B (Roche), 2.5
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mg/ml of pronase (Roche) and 3U/ml of dispase (Sigma-Aldrich) in Ham’s F12/DMEM medium
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(Gibco) for 40 minutes at 37°C and then recovered using a 500-μm Nitex mesh. The tissue was
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further digested by two sequential digestions in trypsin-0.05% EDTA (Gibco) for 15 minutes
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each at 37°C. The cell suspension collected was then filtered through a 250-ȝm Nitex mesh. Cells
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were washed twice, centrifuged at 400 g for 5 min and filtered again through a 100-ȝm Nitex
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mesh. Primary human gastric cells were seeded at a density of 4.105 cells/well in 24-well
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collagen I-coated culture plates (Becton Dickinson). The culture medium consisted of Ham’s
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F12/DMEM (v/v) (Gibco) supplemented with 10% foetal calf serum (FCS; Sigma-Aldrich), 0.5
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ng/ml of epidermal growth factor (EGF; Gibco), 15 ng/ml of hepatocyte growth factor (HGF;
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Miltenyi Biotec), 50 U/ml of penicillin and 50 μg/ml of streptomycin (Gibco). The cultures were 5
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incubated at 37°C in a humidified atmosphere with 5% CO2 before infection assays with H.
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pylori.
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Human gastric cell line culture. AGS cell line (ATCC number: CRL 1739) was cultured
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in 75 cm² flasks in DMEM supplemented with 10% (v/v) FCS, 50 U/ml penicillin and 50 μg/ml
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streptomycin. AGS cells were seeded at a density of 4.105 cells/well in 24-well culture plates.
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The cultures were maintained at 37°C in a humidified atmosphere with 5% CO2.
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Bacterial culture. The H. pylori strains used throughout the study were B128 and
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B128¨cagM. H. pylori B128 strain (cagA, vacA: s1/m2) was isolated from a gastritis patient (15).
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This strain contains an entirely functional cag-PAI. B128¨cagM isogenic mutant was obtained by
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natural transformation, allelic exchanges and insertion of a chloramphenicol resistance cassette
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(28). PCR of the cagM gene empty site was performed to confirm the mutagenesis. Deletion of
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the cagM gene renders H. pylori TIVSS dysfunctional in such a way that B128¨cagM strains
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become unable to perform efficient translocation of CagA or peptidoglycan into epithelial cells
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(28). H. pylori strains were routinely cultured on Skirrow’s medium (Oxoid) and incubated for 48
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h at 37°C in microaerobic conditions using CampyGen bags (Oxoid). For cell infection assays,
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suspensions of H. pylori B128 and B128¨cagM were prepared in the cell culture medium using
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24 h bacterial cultures. Bacteria were added to cells at a multiplicity of infection (MOI) equal to
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10 or 100 bacteria per cell. Bacterial concentrations were determined by measuring the optical
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density of the culture at 600 nm. In addition, colony-forming unit counts of H. pylori were
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performed.
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Cell infection assays with H. pylori. Primary human gastric epithelial cells and AGS
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cells were incubated in Ham’s F12/DMEM culture medium without growth factors, FCS or
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antibiotics for 12 h prior to infection. Cell monolayers were then infected with H. pylori B128 or
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B128¨cagM for 3h or 24h at 37°C in a humidified atmosphere of 5% CO2. Culture supernatants
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were collected, centrifuged (400 g, 5 min, 20°C) and stored at -80°C until used. Cultures without
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bacteria were used as controls. To assess the involvement of epidermal growth factor receptor
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(EGFR) signaling in inflammatory reactions induced by H. pylori, PGEC cells were pretreated for
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2h with tyrphostin AG1478 (2 ȝmol/l; Sigma-Aldrich), a specific inhibitor of EGFR tyrosine
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kinase, before they were infected by H. pylori.
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RNA extraction. Total RNA was extracted from frozen gastric biopsies using MagNA
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Pure Compact system and RNA Isolation Kit (Roche) according to the Manufacturer’s
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instructions. Total RNA extraction from primary epithelial cells and AGS cells was performed
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using the Nucleo-Spin XS RNA extraction kit according to the Manufacturer’s instructions
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(Macherey-Nagel). RNA was eluted in 10 μl of RNAse-free H2O supplemented with 40 units of
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RNaseout® (Invitrogen). RNA concentrations and purity were determined using the Nanodrop
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2000 spectrophotometer (Thermo Scientific) and visualized using 0.8% (w/v) agarose gel
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electrophoresis containing 0.5 ȝg/ml of GelRed® (Fluoroprobes).
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Reverse transcription and Real-time PCR analysis. Total RNA (2 μg) was reverse
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transcribed using SuperScript II kit (Invitrogen) according to the manufacturer’s instructions.
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Quantitative RT-PCR was performed in 96-well plates using LightCycler-FastStart DNA
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MasterPlusSYBR GREEN I kit (Roche) on LightCycler 480 (Roche). Reaction mixture consisted
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of 1X DNA Master Mix (Applied Biosystems), 1 ȝM forward and reverse primers designed using
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Primer 3 software and 12.5 ng of cDNA template in a total volume of 10 μl. PCR conditions were 7
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as follows: 5 min at 95°C, 40 amplification cycles comprising 20 s at 95°C, 15 s at 64°C and 20 s
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at 72°C. Samples were normalized with regard to two independent control housekeeping genes
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(Glyceraldehyde-Phospho-Dehydrogenase and ȕ2-microglobulin) and reported according to the
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ǻǻCT method as RNA fold increase: 2-ǻǻCT= 2-(ǻCT stimulated- ǻCTunstimulated).
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Gastric biopsy samples. Patients with dyspeptic symptoms scheduled for upper
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gastrointestinal endoscopy were prospectively enrolled in the study. The use of gastric tissue
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samples was approved by the local ethics committee (CPP, CHU de Poitiers, protocol #09.10.23).
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Under fully informed consent, 15 cagA-positive gastric biopsies, 15 cagA-negative gastric
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biopsies and 15 normal mucosa biopsies were collected.
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H. pylori detection and determination of cagPAI status. H. pylori detection in human
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gastric biopsies was performed by bacterial culture and Scorpion PCR as previously described
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(16). H. pylori strains from human biopsies were cultured and 24h cultures were used for DNA
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extraction using MagNA Pure Compact system and DNA Isolation Kit (Roche) according to the
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manufacturer’s instructions. A PCR was then performed to determine cagA and cagM status as
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previously described (17, 18). In clinical isolates, the presence of cagA and cagM was used as a
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marker of the presence of cag-PAI.
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Enzyme linked immunosorbent assay. Levels of CXCL1, CXCL5, CXCL8, BD2 and
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BD3 in cell culture supernatants were determined in duplicates using Human ELISA
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development kits (R&D systems for CXCL1 and BD3, and PeproTech for CXCL8, CXCL5 and
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BD2) in accordance with the manufacturers’ specifications.
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Immunocytofluorescence. Primary gastric epithelial cells grown in 24-well plates for
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four days were collected using trypsin-0,05% EDTA and deposited by cytocentrifugation (250 g,
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6 min) on slides at a density of 5.104 cells/slide and then stored at -20°C before completion of 8
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protein expression studies by immunofluorescence. After thawing, cells were fixed in a 4%
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paraformaldehyde/PBS bath, pH 7.4 for 20 min at room temperature. Saturation of nonspecific
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sites was performed for 30 min at room temperature using 5% donkey serum PBS for cytokeratin
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18 (CK18) labeling and 5% goat serum PBS for mucin 1 (MUC1), MUC5AC, MUC6 and trefoil
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factor 1 (TFF-1) labeling. Incubation with specific primary antibodies (Ab) for each antigen was
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then performed in a humid chamber for 2 h at room temperature. The primary mouse monoclonal
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Ab anti-human CK18 and MUC6 (sc-58727 and sc-33668, respectively, Santa Cruz
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Biotechnology) were used at 2 μg/ml in 1% donkey serum PBS, the primary rabbit polyclonal Ab
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anti-human MUC1, MUC5AC and TFF-1 (sc-15333, sc-20118 and sc-28925, respectively; Santa
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Cruz Biotechnology) were used at 4 μg/ml in 1% goat serum PBS. Incubation with donkey anti-
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mouse IgG secondary Ab coupled to Alexa-fluor® 488 (Invitrogen) or goat anti-rabbit IgG
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secondary Ab coupled with Rhodamine-RedX® (Jackson Immunoresearch) was carried out for 45
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min in a dark humid chamber at room temperature. In addition, omission of the first antibody was
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used as a negative control. After successive washes, slides were mounted with cover slips using a
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DAPI mounting medium (Santa Cruz Biotechnology). The slides were then observed by confocal
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microscopy using Olympus Fluoview™ FV1000 microscope (Olympus).
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Statistical analysis. The data presented constitute the average obtained from
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independently performed experiments with a standard error of the mean (SEM). Statistical
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analysis of significance was calculated using Kruskal-Wallis one-way ANOVA analysis of
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variance test followed by Dunn’s comparison between groups. The p values 0.05 were
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considered as significant.
194 195 9
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Results
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Expression of CK18, MUC1, MUC5AC, MUC6 and TFF1 in cultivated PGEC. Preliminary
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evaluation of enzymatic digestion duration, adhesion and culture media conditions was needed in
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order to provide an environment suitable for growing PGEC in vitro. Several filtration and
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washing steps performed between and after digestion were needed so as to remove dissolved
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collagen, scraps and highly viscous mucus that prevented cell adhesion. Proliferation rate was
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highly dependent on the initial cellular density, which was set to 4 x 105 cells at seeding, and rat
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tail collagen I coating was required for cell adhesion. A mix of Ham's F12/DMEM (v/v)
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containing 10% heat-inactivated FCS ensured cell expansion; supplementation with EGF and
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HGF significantly increased the proliferation rate. Within the initial 24h of culture, multiple
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clusters of small flat polygonal cells could be observed. The colonies spread out and formed a
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subconfluent layer three days later and could be maintained in culture for up to eight days. To
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confirm the nature of the cultured primary gastric cells, an immunofluorescence study of the
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CK18 epithelial marker, TFF1 and gastric mucins (MUC1, MUC5AC and MUC6) was
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performed. PGEC and AGS cells stained positive for CK18, MUC1, MUC5AC and TFF1 (figure
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1). PGEC cells stained negative for MUC6 (data not shown). Positive staining for CK18, MUC1,
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MUC5AC and TFF1 in isolated primary cells indicated that they are gastric surface mucous
213
epithelial cells. In addition, levels of MUC1 and TFF1 proteins appeared to be higher in PGEC
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than in AGS cells. This finding was in agreement with MUC1 and TFF1 mRNA expression
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levels detected in both cellular models (data not shown).
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Dose-dependent induction of chemokines, cytokines and antimicrobial peptides in
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PGEC infected with H. pylori. PGEC were infected with H. pylori B128 at MOIs of both 10 and
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100 for 3h and the expression of a panel of cytokines (TNFĮ, IL1-ȕ), chemokines (CXCL1, 10
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CXCL2, CXCL3, CXCL5, CXCL8 and CCL20) and antimicrobial peptides (S100A7, S100A8,
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S100A9, BD1, BD2 and BD3) was then analyzed by RT-QPCR analysis (figure 1S,
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supplementary data). Significant enhanced mRNA expression by PGEC of CXCL1-3, CXCL5,
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CXCL8, CCL20, TNFĮ and BD2 was observed following H. pylori infection at a MOI of 100
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compared to uninfected controls (figure 1S). Under these conditions, IL1-ȕ, S100A7, S100A8,
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S100A9 and BD1 expression were not detected whereas BD3 expression was unchanged (data
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not shown). mRNA expression of CXCL1-3, CXCL5, CXCL8, CCL20, TNFĮ and BD2 after co-
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culture of PGEC with H. pylori at a MOI of 10 was slightly but not significantly upregulated
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(figure 1S).
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An early cagPAI-dependent induction of inflammatory mediators in PGEC. In order
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to establish a transcriptional inflammatory profile comparing primary epithelial cells and AGS
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cells, and also so as to investigate the role of H. pylori’s functional TIVSS in eliciting the
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inflammatory response, the expression of inflammatory mediators induced by H. pylori B128 and
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B128ǻcagM at a MOI of 100 was further compared in PGEC and in AGS cells after 3h of
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infection (figure 2). Whereas stimulation of primary epithelial cells with H. pylori B128 induced
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significant mRNA expression of CXCL1-3, CXCL5, CXCL8, CCL20, TNFĮ and BD2 compared
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to unstimulated controls, no significant induction of any of these mediators was observed after
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stimulation with H. pylori B128ǻcagM (figure 2). However, statistically significant differences
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in inflammatory mediator expression were not observed between infections with both bacterial
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strains. In AGS cells, the H. pylori response profile is pronouncedly different. Among the
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inflammatory mediators, only CXCL5 and CXCL8 mRNA showed significantly higher
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expression compared to uninfected controls following H. pylori B128 infection, while no
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significant induction of any of these mediators was observed after co-culture with H. pylori 11
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B128ǻcagM (figure 2). CCL20 mRNA expression was not detected in AGS cell line upon
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infection. On the other hand, H. pylori-induced mRNA expression of CXCL1-3, TNFĮ and BD2
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was higher in PGEC than in AGS cells. At 24h post infection, both cellular models expressed a
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similar profile of inflammatory mediators as compared to an earlier time of infection (figure 3),
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but cag-PAI-dependent induction of inflammatory mediators was not observed. In addition,
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chemokine expression levels induced by H. pylori were lower than those observed after 3h of
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infection. In contrast, BD2 and BD3 mRNA expression induced by H. pylori was higher at 24h
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post-infection. Significantly enhanced expression of BD2 mRNA was observed in PGEC
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following H. pylori B128 and B128ǻcagM infection (figure 3). In addition, the role of the EGFR
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pathway in chemokine expression of PGEC induced by H. pylori was evaluated using AG1478, a
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specific inhibitor of EGFR tyrosine kinase (figure 2S). This inhibitor reduced CXCL1, 5 and 8
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mRNA expression induced by both bacterial strains at 3h post-infection. AG1478 does not affect
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bacterial viability in the absence of host cells (data not shown). After 24h of infection, inhibition
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of the EGFR had no further impact on chemokine expression (data not shown).
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A cagPAI-independent production of CXCL1, CXCL5 and CXCL8. CXCL1,
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CXCL5, CXCL8, BD2 and BD3 production was quantified by ELISA in PGEC and AGS
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supernatants after 24h of infection with H. pylori B128 or B128ǻcagM at a MOI of 100 (figure
260
4). Production of CXCL1, CXCL5 and CXCL8 was significantly higher in H. pylori B128 and
261
B128ǻcagM-infected PGEC compared to uninfected cells. The amounts of chemokine produced
262
were similar when cells were stimulated with either the wild-type strain or the TIVSS-deficient
263
strain at 24h post-infection. In contrast, significant production of CXCL8 alone was observed in
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AGS cells infected with H. pylori B128 and the protein levels of these three chemokines were 12
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lower than those of primary cells. BD2 and BD3 protein levels could not be detected (below 8
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and 64 pg/ml, respectively) either in PGEC or in AGS culture supernatants.
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Chemokines and BD2 mRNA expression during H. pylori gastritis. CXCL1-3,
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CXCL5, CXCL8 and BD2 mRNA expression in the gastric mucosa of 30 patients infected with
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either H. pylori cagM-positive or cagM-negative strains was examined in order to analyze the
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induction of these mediators according to cagPAI status compared to normal gastric mucosa. All
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the cagM-positive strains were positive for CagA and all the cagM-negative strains were negative
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for cagA. CXCL2, CXCL3, CXCL5, CXCL8 and BD2 mRNA expression were significantly
273
higher in H. pylori cagM-positive mucosa compared to normal mucosa but CXCL1 mRNA levels
274
in non-infected and infected patients were similar (figure 5). In patients infected with cagM-
275
negative strains, only CXCL2, CXCL5 and CXCL8 mRNA expression significantly increased
276
compared to normal gastric mucosa.
277 278
Discussion
279
In this study, a novel and reproducible experimental protocol based on sequential enzymatic
280
digestions was developed to isolate gastric epithelial cells from human stomach samples
281
generated after sleeve gastrectomy. Isolation and expansion of PGEC has always been difficult,
282
partially due to the fragility of these cells and to the lack of sufficient quantities of gastric
283
material. Gastric sleeve operations allow for recovery of the large portions of normal gastric
284
tissue needed to obtain a high cell yield. However, several attempts and optimization steps were
285
needed before it was possible to define and set up culture conditions conducive to growth of the
286
PGEC. Other cellular models, most of them derived from gastric adenocarcinoma such as AGS,
287
KATO-3 and MKN-45 cell lines, had been employed to study the cellular changes triggered by 13
288
H. pylori. However, because of their tumoral origin, they may not be considered as the bona fide
289
counterparts of the normal gastric epithelial cells required in study of H. pylori response. The
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PGEC obtained with our protocol were mucus-secreting cells as shown by immunodetection of
291
MUC1, MUC5AC and TFF-1, highly glycosylated proteins specifically present in mucous cells
292
of the antrum and the fundus (19, 20). PGEC stained negative for MUC6, of which the expression
293
is known to be localized in the lower part of the gastric pits (21). It was shown that gastric
294
epithelial cells specifically express only one of the MUC5AC and MUC6 mucins, and that H.
295
pylori is very closely associated with epithelial cells that produce MUC5AC (21). These findings
296
would suggest that PGEC are relevant for study of H. pylori infection. Compared to AGS cells,
297
PGEC expressed higher level of the proteins MUC1 and TFF1, both of which play an important
298
role in regulation of inflammatory signaling (22, 23). Most notably, it was shown that MUC1
299
regulates CXCL8 production by gastric epithelial cells in response to H. pylori (22). The aim of
300
this work was to provide a comparative inflammatory profile study involving PGEC and AGS
301
cells after H. pylori infection.
302
Induction by H. pylori of PGEC expression of CXCL1-3, CXCL5, CXCL8, CCL20,
303
TNFĮ and BD2 was more pronounced with the wild-type strain than with CagM mutant at 3h
304
post-infection whereas this effect was not observed after 24h of infection. This finding suggests
305
that H. pylori TIVSS plays a major role in the early induction of these inflammatory mediators
306
during infection. In AGS cells, significant induction of CXCL5 and CXCL8 mRNA expression
307
was the only induction observed following 3h of stimulation with H. pylori B128. These results
308
suggest that PGEC were more reactive to H. pylori cag-PAI than AGS cells. However,
309
production by PGEC of similar levels of CXCL1, CXCL5 and CXCL8 following stimulation
310
with H. pylori B128 and B128ǻcagM at 24h post-infection suggest that other virulence factors 14
311
are involved in chemokines production, and that they compensate for the early mRNA induction
312
specifically associated with functional cag-PAI. The similar levels of chemokines mRNA
313
induced by both H. pylori strains after 24h of stimulation support this hypothesis. In addition,
314
results of EGFR inhibition in PGEC show that the EGFR pathway is involved in chemokine
315
induction by both H. pylori strains, suggesting a cag-PAI-independent mechanism. In agreement,
316
we showed that CXCL5 and CXCL8 mRNA expression was significantly higher in the gastric
317
mucosa of patients infected with H. pylori compared to normal mucosa, but no significant
318
difference was observed between patients infected with cagA-positive strains and those infected
319
with cagA-negative strains. CXCL1, 2, 3, 5 and 8 proteins are neutrophil-activating chemokines
320
contributing to establishment of a chemotactic gradient during inflammation, and CCL20 plays
321
an important role in the homing of lymphocytes and dendritic cells to sites of inflammation.
322
Infection with H. pylori induces production of a large panel of chemokines by PGEC that leads to
323
infiltration towards the gastric mucosa of inflammatory cells involved in the pathophysiological
324
process of the infection. Thus, these cells can allow the development of relevant ex vivo co-
325
culture models, for example with dendritic cells or lymphocytes, as they express CCL20 in
326
response to H. pylori infection.
327
CXCL8 expression during H. pylori infection has long been described as being directly
328
linked to CagA translocation into epithelial cells (24, 25). Actually, CagA is not essential for
329
induction of this chemokine since CagA-deficient strains of H. pylori can likewise induce CXCL8
330
production by several cell lines at levels similar to those of wild-type strains (1, 26). In fact, it is
331
the presence of a functional TIVSS that plays a major role in the induction of CXCL8 and other
332
proinflammatory cytokines during H. pylori infection through NF-țB or AP-1 activation (27).
333
Several cag-PAI-dependent but cagA-independent NF-țB activation mechanisms have recently 15
334
been suggested, involving at least six different signaling pathways (27). One of them is the
335
internal recognition of translocated PG through TIVSS by the Nod1 protein, which activates NF-
336
țB signaling and thereby induces CXCL8 production (28). An alternative explanation for
337
chemokine production by gastric epithelial cells stimulated by B128ǻcagM strain is that since
338
TIVSS promotes intimate interactions between the bacteria and epithelial cells, endocytosis of
339
some H. pylori products, such as PG, is facilitated. This hypothesis was established by Viala et
340
al. to explain detection of radioactively labeled PG inside AGS cells exposed to ǻcagM-H. pylori
341
mutants (28). Furthermore, it seems that H. pylori can ensure PG translocation inside host cells
342
through the outer membrane vesicles that are constantly released by the bacterium (29). Recently,
343
it was suggested that CagL, via interactions with host integrins, can trigger pro-inflammatory
344
responses independently of CagA translocation or NOD1 signaling (30). As a result, the T4SS
345
apparatus per se could elicit host proinflammatory responses independently of its substrates (30).
346
Finally, chemokine production during H. pylori infection could also be induced by other bacterial
347
virulence factors. For example, Beswick et al. showed that the interaction of urease with the
348
surface receptor CD74 can activate NF-țB, which induces CXCL8 production (31). Smith et al.
349
reported that TLR2 and TLR5 are required for H. pylori-induced NF-țB activation and
350
chemokine expression by epithelial cells (5). It has also been suggested that H. pylori-induced
351
expression of TLR2 and TLR5 can qualitatively shift cag-PAI-dependent to cag-PAI-
352
independent pro-inflammatory signaling pathways leading to CXCL8 production in HEK293
353
cells transfected with TLR2 and TLR5 (32). The TLR2 ligand of H. pylori is controversial.
354
Indeed, it has also been suggested that lipopolysaccharide functions as a TLR2 ligand and
355
induces CXCL1-3 and CCL20 expression in MKN45 cells (33). Yet another study suggested that
356
H. pylori LPS does not activate TLR2 even at high concentrations of LPS (34). On the other 16
357
hand, H. pylori-heat shock protein 60 has been reported to induce CXCL8 via a TLR2 pathway in
358
monocytes (35). In addition, EGFR seems to play an important role in inflammatory reaction of
359
PGEC; this pathway can be activated by H. pylori independently of the presence of a functional
360
cag-PAI, as previously reported (36). Our data suggest that gastric epithelial cells can secrete
361
chemokines via cag-PAI-independent pathways, which have been shown to play a major role in
362
H. pylori-induced delayed inflammatory response.
363
Furthermore, expression of antimicrobial peptides in response to H. pylori has been
364
investigated as part of innate response and defense upon infection. Early cag-PAI-dependent BD2
365
mRNA expression was observed in PGEC infected with H. pylori whereas no significant
366
induction of this peptide was observed in AGS cells. In agreement, BD2 mRNA expression was
367
higher in the gastric mucosa of patients infected with cagA-positive strains than in those infected
368
with cagA-negative strains, a finding suggesting cag-PAI-dependent induction of this peptide.
369
BD2 was shown to be induced in the gastric epithelial tissues during H. pylori infection (37).
370
Furthermore, cag-PAI-dependent induction of BD2 was also observed in MKN45 cells infected
371
with H. pylori (38). The binding of BD2 on the surface of H. pylori cells was recently shown
372
(39), and it is a phenomenon that may explain the absence of detection of BD2 proteins in the cell
373
culture supernatants. Our results support the view that BD2 is expressed in response to early and
374
chronic H. pylori infection and that cag-PAI is involved in this antimicrobial peptide mRNA
375
induction.
376
Taken together, this new culture model of primary human gastric epithelial cells using
377
pieces of stomach enabled development of an H. pylori infection model and characterization of
378
the inflammatory profile generated upon infection. The cellular response of PGEC was
379
reproducible despite the different genetic backgrounds of the patients included in the study. 17
380
Furthermore, the inflammatory response of PGEC upon infection with H. pylori is closer to the in
381
vivo response characteristic of infected gastric mucosa than was that of previously described cell
382
lines, of which the response could be altered due to their tumoral physiology. The model also
383
supported the hypothesis of involvement of H. pylori TIVSS in eliciting an inflammatory
384
response at early phases of infection. At a later phase, our data supported the view that
385
inflammatory mediator expression and production are largely due to cag-PAI substrate
386
independent virulence factors. This largely novel human gastric epithelial cell model could be
387
used to investigate the interactions of stomach epithelium with inflammatory cells and to study
388
other functional aspects of infection with H. pylori in order to better characterize the pathological
389
processes associated with gastric infections.
390 391
Acknowledgements
392
This study was supported in part by a grant from the French Ministry of Health (PHRC
393
Pylorikine). Work in Ivo G Boneca laboratory was supported by ERC starting Grant
394
(PGNfromSHAPEtoVIR n°202283). We thank Emilie Peyrot and Damien Chassaing for
395
technical assistance and Jeffrey Arsham for English revision of the paper. There are no conflicts
396
of interest to disclose.
397 398
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399
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525
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527 528
Figure legends
529 530
Figure 1. Immunodetection of CK18, MUC1, MUC5AC and TFF1 in PGEC cells and in AGS
531
cells. Scale bar: 50 μm.
532 533
Figure 2. Gene expression of PEGC and AGS cells stimulated by H. pylori. Cells were infected
534
by H. pylori B128 or B128ǻcagM at a MOI of 100 for 3h. QRT-PCR analysis was carried out on 24
535
total RNA of independent cultures from 5 different patients. mRNA expression levels are
536
expressed as the fold increase above unstimulated cultures. Data are represented as mean + SEM.
537
Statistically significant differences in inflammatory mediator production were studied between
538
infected cells and uninfected cells as well as between cells stimulated with H. pylori B128 and
539
B128ǻcagM. *p< 0.05 and **p
1
Chemokines and antimicrobial peptides cag-dependent early response to
2
Helicobacter pylori infection in primary human gastric epithelial cells
3
Pascale Mustapha1, Isabelle Paris1, 2, Magali Garcia1, 2, Cong Tri Tran1, 2, Julie Cremniter1, 2,
4
Martine Garnier1, 2, Jean-Pierre Faure2, Thierry Barthes3, Ivo G. Boneca4, 5, Franck Morel1, Jean-
5
Claude Lecron1, 2, Christophe Burucoa1, 2, Charles Bodet1#.
6
7
1. Laboratoire Inflammation, Tissus Epithéliaux et Cytokines (LITEC - EA 4331), Université de
8
Poitiers, France.
9
2. Centre Hospitalier Universitaire de Poitiers, France.
10
3. Polyclinique de Poitiers, France.
11
4. Institut Pasteur, Unité de Biologie et Génétique de la Paroi Bactérienne, Paris, France.
12
5. INSERM, Equipe Avenir, Paris, France.
13
# Address correspondence to Charles Bodet, [email protected]
14
Running Head: Primary gastric cell response to H. pylori
15
1
16
Abstract
17
Helicobacter pylori infection systematically causes chronic gastric inflammation that can persist
18
asymptomatically or evolve towards more severe gastroduodenal pathologies such as ulcer,
19
MALT lymphoma or gastric cancer. The cag pathogenicity island (cag-PAI) of H. pylori allows
20
for translocation of the virulent protein CagA and fragments of peptidoglycan into host cells,
21
thereby inducing production of chemokines, cytokines and antimicrobial peptides. In order to
22
characterize inflammatory response to H. pylori, a new experimental protocol for isolating and
23
culturing primary human gastric epithelial cells was established using pieces of stomach from
24
patients who had undergone sleeve gastrectomy. Isolated cells expressed markers indicating that
25
they were mucin-secreting epithelial cells. Challenge of primary epithelial cells with H. pylori
26
B128 underscored early dose-dependent induction of mRNA expression of the inflammatory
27
mediators CXCL1-3, CXCL5, CXCL8, CCL20, BD2 and TNFĮ. In AGS cells, significant
28
expression of only CXCL5 and CXCL8 was observed following infection suggesting that these
29
cells were less reactive than primary epithelial cells. Infection of both cellular models with H.
30
pylori B128ǻcagM, a cag-PAI mutant, resulted in weak inflammatory mediator mRNA
31
induction. At 24h post-infection of primary epithelial cells with H. pylori, inflammatory mediator
32
production was largely due to cag-PAI-substrate-independent virulence factors. Thus, H. pylori
33
cag-PAI substrate appears to be involved in eliciting an epithelial response during the early
34
phases of infection. Afterwards, other virulence factors of the bacterium take over in
35
development of the inflammatory response. Using a relevant cellular model, this study provides
36
new information on the modulation of inflammation during H. pylori infection.
37 38 2
39
Introduction
40
It is now widely established that infection with Helicobacter pylori is the leading cause of
41
chronic gastritis, duodenal ulcers and gastric cancer. This Gram-negative bacterial pathogen
42
selectively colonizes the human gastric epithelium, thereby inducing an inflammatory response
43
characterized by massive neutrophil infiltration and large-scale production of cytokines and
44
chemokines, mainly interleukin (IL)-8 (CXCL8).
45
The cag Pathogenicity Island (cag-PAI) is one of the most widely studied virulence
46
factors of H. pylori. This 37 kb DNA segment encodes a type IV secretion apparatus (TIVSS)
47
that delivers virulent factors such as the CagA protein as well as fragments of peptidoglycan (PG)
48
into host cells (1-3). CagM is a 43.7 kDa protein, which is also encoded by cag-PAI. Along with
49
CagX and CagT, CagM forms an outer membrane-associated TIVSS sub-complex (4).
50
Systematic mutagenesis of cag-PAI genes showed that H. pylori ǻcagM strains cannot perform
51
efficient translocation of CagA or PG into epithelial cells (3). CagA and CagM are consequently
52
important when assessing the virulence of H. pylori strains.
53
In order to study the interactions between cag-PAI and epithelial cells, several cell line
54
models derived from gastric adenocarcinoma such as the AGS or KATO-3 cell lines have been
55
used. Even though these models have clarified many aspects of the changes triggered by H. pylori
56
at the cellular level such as establishment of the inflammatory response, at a later time these cell
57
lines were found to have lost some of the key elements in signaling pathways involved in cellular
58
responses to H. pylori infection. For example, the AGS cell line lacks the Toll-like Receptor 2
59
(TLR2) and the MD2 cofactor, which is essential for lipopolysaccharide (LPS) recognition by
60
TLR4 (5).
3
61
As a result, so as to obtain a cellular model more relevant to define and characterize the
62
pathophysiological processes associated with H. pylori infection, development of primary human
63
epithelial cell models was considered necessary. Even though primary culture of human gastric
64
epithelial cells has been limited, mainly on account of the unavailability of sufficient quantities of
65
human gastric tissue and the difficulty of isolating and expanding these cells, a few models have
66
been developed using organ donors (6), fetal tissue (7) and gastric biopsies (8). Isolation of
67
gastric epithelial cells from organ donors was used to study the characteristics of attachment of
68
H. pylori to cultured cells (9) and its effects on gastrin secretion (10). In another study, Basque et
69
al. developed a primary epithelial cell model from legally aborted fetuses (7), and it was used to
70
study the secretion of pepsinogen and gastric lipase (11) and to examine the effects of growth
71
factors on cell proliferation and regeneration (12). In a third approach, Smoot et al. developed a
72
protocol for primary culture of epithelial cells using gastric biopsies (8). Using the same model,
73
Bäckhed et al. reported that H. pylori infection of primary cells induced a regulated production of
74
IL-6, IL-8, and tumor necrosis factor–Į, whereas infection of cell lines only resulted in IL-8
75
production (13). A CagA-dependent induction of cytoskeletal rearrangements and alterations in
76
cell junctions was likewise reported with regard to this model (14). However, given the low yield
77
in primary cells, these partial results needed to be complemented by parallel studies on cell lines.
78
And even though the aforementioned models have clearly provided valuable insights into the
79
functioning of normal gastric epithelial cells, up until now the inflammatory response associated
80
with H. pylori has remained poorly investigated in primary cells. The aim of this study is to
81
characterize H. pylori-associated inflammatory reactions by applying a new protocol allowing for
82
isolation and culture of human primary gastric epithelial cells (PGEC) using normal gastric tissue
83
obtained from obese subjects having undergone gastric sleeve surgery. Isolation of PGEC from 4
84
large pieces of human stomach is an original approach that has contributed to the development of
85
a pertinent in vitro cell model facilitating study of the expression of the antimicrobial peptides,
86
chemokines and cytokines induced by H. pylori.
87 88
Materials and Methods
89
Human stomach samples. The use of stomach samples for this study was approved by the ethics
90
committee of Poitiers hospital. Under fully informed consent, normal human antra were obtained
91
from H. pylori-negative subjects who had undergone gastric sleeve surgery.
92
Isolation and culture of PGEC from human stomach. Pieces of antrum were
93
abundantly washed with phosphate buffered saline solution (PBS) (Gibco) to remove blood and
94
mucous and then cut along the surgical crease. Fat and muscle layers were discarded. The mucosa
95
was carefully dissected from underlying submucosa and minced into small fragments of about 2
96
mm3 using scalpel blades. Tissues were digested in 0.5 mg/ml of collagenase B (Roche), 2.5
97
mg/ml of pronase (Roche) and 3U/ml of dispase (Sigma-Aldrich) in Ham’s F12/DMEM medium
98
(Gibco) for 40 minutes at 37°C and then recovered using a 500-μm Nitex mesh. The tissue was
99
further digested by two sequential digestions in trypsin-0.05% EDTA (Gibco) for 15 minutes
100
each at 37°C. The cell suspension collected was then filtered through a 250-ȝm Nitex mesh. Cells
101
were washed twice, centrifuged at 400 g for 5 min and filtered again through a 100-ȝm Nitex
102
mesh. Primary human gastric cells were seeded at a density of 4.105 cells/well in 24-well
103
collagen I-coated culture plates (Becton Dickinson). The culture medium consisted of Ham’s
104
F12/DMEM (v/v) (Gibco) supplemented with 10% foetal calf serum (FCS; Sigma-Aldrich), 0.5
105
ng/ml of epidermal growth factor (EGF; Gibco), 15 ng/ml of hepatocyte growth factor (HGF;
106
Miltenyi Biotec), 50 U/ml of penicillin and 50 μg/ml of streptomycin (Gibco). The cultures were 5
107
incubated at 37°C in a humidified atmosphere with 5% CO2 before infection assays with H.
108
pylori.
109
Human gastric cell line culture. AGS cell line (ATCC number: CRL 1739) was cultured
110
in 75 cm² flasks in DMEM supplemented with 10% (v/v) FCS, 50 U/ml penicillin and 50 μg/ml
111
streptomycin. AGS cells were seeded at a density of 4.105 cells/well in 24-well culture plates.
112
The cultures were maintained at 37°C in a humidified atmosphere with 5% CO2.
113
Bacterial culture. The H. pylori strains used throughout the study were B128 and
114
B128¨cagM. H. pylori B128 strain (cagA, vacA: s1/m2) was isolated from a gastritis patient (15).
115
This strain contains an entirely functional cag-PAI. B128¨cagM isogenic mutant was obtained by
116
natural transformation, allelic exchanges and insertion of a chloramphenicol resistance cassette
117
(28). PCR of the cagM gene empty site was performed to confirm the mutagenesis. Deletion of
118
the cagM gene renders H. pylori TIVSS dysfunctional in such a way that B128¨cagM strains
119
become unable to perform efficient translocation of CagA or peptidoglycan into epithelial cells
120
(28). H. pylori strains were routinely cultured on Skirrow’s medium (Oxoid) and incubated for 48
121
h at 37°C in microaerobic conditions using CampyGen bags (Oxoid). For cell infection assays,
122
suspensions of H. pylori B128 and B128¨cagM were prepared in the cell culture medium using
123
24 h bacterial cultures. Bacteria were added to cells at a multiplicity of infection (MOI) equal to
124
10 or 100 bacteria per cell. Bacterial concentrations were determined by measuring the optical
125
density of the culture at 600 nm. In addition, colony-forming unit counts of H. pylori were
126
performed.
6
127
Cell infection assays with H. pylori. Primary human gastric epithelial cells and AGS
128
cells were incubated in Ham’s F12/DMEM culture medium without growth factors, FCS or
129
antibiotics for 12 h prior to infection. Cell monolayers were then infected with H. pylori B128 or
130
B128¨cagM for 3h or 24h at 37°C in a humidified atmosphere of 5% CO2. Culture supernatants
131
were collected, centrifuged (400 g, 5 min, 20°C) and stored at -80°C until used. Cultures without
132
bacteria were used as controls. To assess the involvement of epidermal growth factor receptor
133
(EGFR) signaling in inflammatory reactions induced by H. pylori, PGEC cells were pretreated for
134
2h with tyrphostin AG1478 (2 ȝmol/l; Sigma-Aldrich), a specific inhibitor of EGFR tyrosine
135
kinase, before they were infected by H. pylori.
136
RNA extraction. Total RNA was extracted from frozen gastric biopsies using MagNA
137
Pure Compact system and RNA Isolation Kit (Roche) according to the Manufacturer’s
138
instructions. Total RNA extraction from primary epithelial cells and AGS cells was performed
139
using the Nucleo-Spin XS RNA extraction kit according to the Manufacturer’s instructions
140
(Macherey-Nagel). RNA was eluted in 10 μl of RNAse-free H2O supplemented with 40 units of
141
RNaseout® (Invitrogen). RNA concentrations and purity were determined using the Nanodrop
142
2000 spectrophotometer (Thermo Scientific) and visualized using 0.8% (w/v) agarose gel
143
electrophoresis containing 0.5 ȝg/ml of GelRed® (Fluoroprobes).
144
Reverse transcription and Real-time PCR analysis. Total RNA (2 μg) was reverse
145
transcribed using SuperScript II kit (Invitrogen) according to the manufacturer’s instructions.
146
Quantitative RT-PCR was performed in 96-well plates using LightCycler-FastStart DNA
147
MasterPlusSYBR GREEN I kit (Roche) on LightCycler 480 (Roche). Reaction mixture consisted
148
of 1X DNA Master Mix (Applied Biosystems), 1 ȝM forward and reverse primers designed using
149
Primer 3 software and 12.5 ng of cDNA template in a total volume of 10 μl. PCR conditions were 7
150
as follows: 5 min at 95°C, 40 amplification cycles comprising 20 s at 95°C, 15 s at 64°C and 20 s
151
at 72°C. Samples were normalized with regard to two independent control housekeeping genes
152
(Glyceraldehyde-Phospho-Dehydrogenase and ȕ2-microglobulin) and reported according to the
153
ǻǻCT method as RNA fold increase: 2-ǻǻCT= 2-(ǻCT stimulated- ǻCTunstimulated).
154
Gastric biopsy samples. Patients with dyspeptic symptoms scheduled for upper
155
gastrointestinal endoscopy were prospectively enrolled in the study. The use of gastric tissue
156
samples was approved by the local ethics committee (CPP, CHU de Poitiers, protocol #09.10.23).
157
Under fully informed consent, 15 cagA-positive gastric biopsies, 15 cagA-negative gastric
158
biopsies and 15 normal mucosa biopsies were collected.
159
H. pylori detection and determination of cagPAI status. H. pylori detection in human
160
gastric biopsies was performed by bacterial culture and Scorpion PCR as previously described
161
(16). H. pylori strains from human biopsies were cultured and 24h cultures were used for DNA
162
extraction using MagNA Pure Compact system and DNA Isolation Kit (Roche) according to the
163
manufacturer’s instructions. A PCR was then performed to determine cagA and cagM status as
164
previously described (17, 18). In clinical isolates, the presence of cagA and cagM was used as a
165
marker of the presence of cag-PAI.
166
Enzyme linked immunosorbent assay. Levels of CXCL1, CXCL5, CXCL8, BD2 and
167
BD3 in cell culture supernatants were determined in duplicates using Human ELISA
168
development kits (R&D systems for CXCL1 and BD3, and PeproTech for CXCL8, CXCL5 and
169
BD2) in accordance with the manufacturers’ specifications.
170
Immunocytofluorescence. Primary gastric epithelial cells grown in 24-well plates for
171
four days were collected using trypsin-0,05% EDTA and deposited by cytocentrifugation (250 g,
172
6 min) on slides at a density of 5.104 cells/slide and then stored at -20°C before completion of 8
173
protein expression studies by immunofluorescence. After thawing, cells were fixed in a 4%
174
paraformaldehyde/PBS bath, pH 7.4 for 20 min at room temperature. Saturation of nonspecific
175
sites was performed for 30 min at room temperature using 5% donkey serum PBS for cytokeratin
176
18 (CK18) labeling and 5% goat serum PBS for mucin 1 (MUC1), MUC5AC, MUC6 and trefoil
177
factor 1 (TFF-1) labeling. Incubation with specific primary antibodies (Ab) for each antigen was
178
then performed in a humid chamber for 2 h at room temperature. The primary mouse monoclonal
179
Ab anti-human CK18 and MUC6 (sc-58727 and sc-33668, respectively, Santa Cruz
180
Biotechnology) were used at 2 μg/ml in 1% donkey serum PBS, the primary rabbit polyclonal Ab
181
anti-human MUC1, MUC5AC and TFF-1 (sc-15333, sc-20118 and sc-28925, respectively; Santa
182
Cruz Biotechnology) were used at 4 μg/ml in 1% goat serum PBS. Incubation with donkey anti-
183
mouse IgG secondary Ab coupled to Alexa-fluor® 488 (Invitrogen) or goat anti-rabbit IgG
184
secondary Ab coupled with Rhodamine-RedX® (Jackson Immunoresearch) was carried out for 45
185
min in a dark humid chamber at room temperature. In addition, omission of the first antibody was
186
used as a negative control. After successive washes, slides were mounted with cover slips using a
187
DAPI mounting medium (Santa Cruz Biotechnology). The slides were then observed by confocal
188
microscopy using Olympus Fluoview™ FV1000 microscope (Olympus).
189
Statistical analysis. The data presented constitute the average obtained from
190
independently performed experiments with a standard error of the mean (SEM). Statistical
191
analysis of significance was calculated using Kruskal-Wallis one-way ANOVA analysis of
192
variance test followed by Dunn’s comparison between groups. The p values 0.05 were
193
considered as significant.
194 195 9
196
Results
197
Expression of CK18, MUC1, MUC5AC, MUC6 and TFF1 in cultivated PGEC. Preliminary
198
evaluation of enzymatic digestion duration, adhesion and culture media conditions was needed in
199
order to provide an environment suitable for growing PGEC in vitro. Several filtration and
200
washing steps performed between and after digestion were needed so as to remove dissolved
201
collagen, scraps and highly viscous mucus that prevented cell adhesion. Proliferation rate was
202
highly dependent on the initial cellular density, which was set to 4 x 105 cells at seeding, and rat
203
tail collagen I coating was required for cell adhesion. A mix of Ham's F12/DMEM (v/v)
204
containing 10% heat-inactivated FCS ensured cell expansion; supplementation with EGF and
205
HGF significantly increased the proliferation rate. Within the initial 24h of culture, multiple
206
clusters of small flat polygonal cells could be observed. The colonies spread out and formed a
207
subconfluent layer three days later and could be maintained in culture for up to eight days. To
208
confirm the nature of the cultured primary gastric cells, an immunofluorescence study of the
209
CK18 epithelial marker, TFF1 and gastric mucins (MUC1, MUC5AC and MUC6) was
210
performed. PGEC and AGS cells stained positive for CK18, MUC1, MUC5AC and TFF1 (figure
211
1). PGEC cells stained negative for MUC6 (data not shown). Positive staining for CK18, MUC1,
212
MUC5AC and TFF1 in isolated primary cells indicated that they are gastric surface mucous
213
epithelial cells. In addition, levels of MUC1 and TFF1 proteins appeared to be higher in PGEC
214
than in AGS cells. This finding was in agreement with MUC1 and TFF1 mRNA expression
215
levels detected in both cellular models (data not shown).
216
Dose-dependent induction of chemokines, cytokines and antimicrobial peptides in
217
PGEC infected with H. pylori. PGEC were infected with H. pylori B128 at MOIs of both 10 and
218
100 for 3h and the expression of a panel of cytokines (TNFĮ, IL1-ȕ), chemokines (CXCL1, 10
219
CXCL2, CXCL3, CXCL5, CXCL8 and CCL20) and antimicrobial peptides (S100A7, S100A8,
220
S100A9, BD1, BD2 and BD3) was then analyzed by RT-QPCR analysis (figure 1S,
221
supplementary data). Significant enhanced mRNA expression by PGEC of CXCL1-3, CXCL5,
222
CXCL8, CCL20, TNFĮ and BD2 was observed following H. pylori infection at a MOI of 100
223
compared to uninfected controls (figure 1S). Under these conditions, IL1-ȕ, S100A7, S100A8,
224
S100A9 and BD1 expression were not detected whereas BD3 expression was unchanged (data
225
not shown). mRNA expression of CXCL1-3, CXCL5, CXCL8, CCL20, TNFĮ and BD2 after co-
226
culture of PGEC with H. pylori at a MOI of 10 was slightly but not significantly upregulated
227
(figure 1S).
228
An early cagPAI-dependent induction of inflammatory mediators in PGEC. In order
229
to establish a transcriptional inflammatory profile comparing primary epithelial cells and AGS
230
cells, and also so as to investigate the role of H. pylori’s functional TIVSS in eliciting the
231
inflammatory response, the expression of inflammatory mediators induced by H. pylori B128 and
232
B128ǻcagM at a MOI of 100 was further compared in PGEC and in AGS cells after 3h of
233
infection (figure 2). Whereas stimulation of primary epithelial cells with H. pylori B128 induced
234
significant mRNA expression of CXCL1-3, CXCL5, CXCL8, CCL20, TNFĮ and BD2 compared
235
to unstimulated controls, no significant induction of any of these mediators was observed after
236
stimulation with H. pylori B128ǻcagM (figure 2). However, statistically significant differences
237
in inflammatory mediator expression were not observed between infections with both bacterial
238
strains. In AGS cells, the H. pylori response profile is pronouncedly different. Among the
239
inflammatory mediators, only CXCL5 and CXCL8 mRNA showed significantly higher
240
expression compared to uninfected controls following H. pylori B128 infection, while no
241
significant induction of any of these mediators was observed after co-culture with H. pylori 11
242
B128ǻcagM (figure 2). CCL20 mRNA expression was not detected in AGS cell line upon
243
infection. On the other hand, H. pylori-induced mRNA expression of CXCL1-3, TNFĮ and BD2
244
was higher in PGEC than in AGS cells. At 24h post infection, both cellular models expressed a
245
similar profile of inflammatory mediators as compared to an earlier time of infection (figure 3),
246
but cag-PAI-dependent induction of inflammatory mediators was not observed. In addition,
247
chemokine expression levels induced by H. pylori were lower than those observed after 3h of
248
infection. In contrast, BD2 and BD3 mRNA expression induced by H. pylori was higher at 24h
249
post-infection. Significantly enhanced expression of BD2 mRNA was observed in PGEC
250
following H. pylori B128 and B128ǻcagM infection (figure 3). In addition, the role of the EGFR
251
pathway in chemokine expression of PGEC induced by H. pylori was evaluated using AG1478, a
252
specific inhibitor of EGFR tyrosine kinase (figure 2S). This inhibitor reduced CXCL1, 5 and 8
253
mRNA expression induced by both bacterial strains at 3h post-infection. AG1478 does not affect
254
bacterial viability in the absence of host cells (data not shown). After 24h of infection, inhibition
255
of the EGFR had no further impact on chemokine expression (data not shown).
256 257
A cagPAI-independent production of CXCL1, CXCL5 and CXCL8. CXCL1,
258
CXCL5, CXCL8, BD2 and BD3 production was quantified by ELISA in PGEC and AGS
259
supernatants after 24h of infection with H. pylori B128 or B128ǻcagM at a MOI of 100 (figure
260
4). Production of CXCL1, CXCL5 and CXCL8 was significantly higher in H. pylori B128 and
261
B128ǻcagM-infected PGEC compared to uninfected cells. The amounts of chemokine produced
262
were similar when cells were stimulated with either the wild-type strain or the TIVSS-deficient
263
strain at 24h post-infection. In contrast, significant production of CXCL8 alone was observed in
264
AGS cells infected with H. pylori B128 and the protein levels of these three chemokines were 12
265
lower than those of primary cells. BD2 and BD3 protein levels could not be detected (below 8
266
and 64 pg/ml, respectively) either in PGEC or in AGS culture supernatants.
267
Chemokines and BD2 mRNA expression during H. pylori gastritis. CXCL1-3,
268
CXCL5, CXCL8 and BD2 mRNA expression in the gastric mucosa of 30 patients infected with
269
either H. pylori cagM-positive or cagM-negative strains was examined in order to analyze the
270
induction of these mediators according to cagPAI status compared to normal gastric mucosa. All
271
the cagM-positive strains were positive for CagA and all the cagM-negative strains were negative
272
for cagA. CXCL2, CXCL3, CXCL5, CXCL8 and BD2 mRNA expression were significantly
273
higher in H. pylori cagM-positive mucosa compared to normal mucosa but CXCL1 mRNA levels
274
in non-infected and infected patients were similar (figure 5). In patients infected with cagM-
275
negative strains, only CXCL2, CXCL5 and CXCL8 mRNA expression significantly increased
276
compared to normal gastric mucosa.
277 278
Discussion
279
In this study, a novel and reproducible experimental protocol based on sequential enzymatic
280
digestions was developed to isolate gastric epithelial cells from human stomach samples
281
generated after sleeve gastrectomy. Isolation and expansion of PGEC has always been difficult,
282
partially due to the fragility of these cells and to the lack of sufficient quantities of gastric
283
material. Gastric sleeve operations allow for recovery of the large portions of normal gastric
284
tissue needed to obtain a high cell yield. However, several attempts and optimization steps were
285
needed before it was possible to define and set up culture conditions conducive to growth of the
286
PGEC. Other cellular models, most of them derived from gastric adenocarcinoma such as AGS,
287
KATO-3 and MKN-45 cell lines, had been employed to study the cellular changes triggered by 13
288
H. pylori. However, because of their tumoral origin, they may not be considered as the bona fide
289
counterparts of the normal gastric epithelial cells required in study of H. pylori response. The
290
PGEC obtained with our protocol were mucus-secreting cells as shown by immunodetection of
291
MUC1, MUC5AC and TFF-1, highly glycosylated proteins specifically present in mucous cells
292
of the antrum and the fundus (19, 20). PGEC stained negative for MUC6, of which the expression
293
is known to be localized in the lower part of the gastric pits (21). It was shown that gastric
294
epithelial cells specifically express only one of the MUC5AC and MUC6 mucins, and that H.
295
pylori is very closely associated with epithelial cells that produce MUC5AC (21). These findings
296
would suggest that PGEC are relevant for study of H. pylori infection. Compared to AGS cells,
297
PGEC expressed higher level of the proteins MUC1 and TFF1, both of which play an important
298
role in regulation of inflammatory signaling (22, 23). Most notably, it was shown that MUC1
299
regulates CXCL8 production by gastric epithelial cells in response to H. pylori (22). The aim of
300
this work was to provide a comparative inflammatory profile study involving PGEC and AGS
301
cells after H. pylori infection.
302
Induction by H. pylori of PGEC expression of CXCL1-3, CXCL5, CXCL8, CCL20,
303
TNFĮ and BD2 was more pronounced with the wild-type strain than with CagM mutant at 3h
304
post-infection whereas this effect was not observed after 24h of infection. This finding suggests
305
that H. pylori TIVSS plays a major role in the early induction of these inflammatory mediators
306
during infection. In AGS cells, significant induction of CXCL5 and CXCL8 mRNA expression
307
was the only induction observed following 3h of stimulation with H. pylori B128. These results
308
suggest that PGEC were more reactive to H. pylori cag-PAI than AGS cells. However,
309
production by PGEC of similar levels of CXCL1, CXCL5 and CXCL8 following stimulation
310
with H. pylori B128 and B128ǻcagM at 24h post-infection suggest that other virulence factors 14
311
are involved in chemokines production, and that they compensate for the early mRNA induction
312
specifically associated with functional cag-PAI. The similar levels of chemokines mRNA
313
induced by both H. pylori strains after 24h of stimulation support this hypothesis. In addition,
314
results of EGFR inhibition in PGEC show that the EGFR pathway is involved in chemokine
315
induction by both H. pylori strains, suggesting a cag-PAI-independent mechanism. In agreement,
316
we showed that CXCL5 and CXCL8 mRNA expression was significantly higher in the gastric
317
mucosa of patients infected with H. pylori compared to normal mucosa, but no significant
318
difference was observed between patients infected with cagA-positive strains and those infected
319
with cagA-negative strains. CXCL1, 2, 3, 5 and 8 proteins are neutrophil-activating chemokines
320
contributing to establishment of a chemotactic gradient during inflammation, and CCL20 plays
321
an important role in the homing of lymphocytes and dendritic cells to sites of inflammation.
322
Infection with H. pylori induces production of a large panel of chemokines by PGEC that leads to
323
infiltration towards the gastric mucosa of inflammatory cells involved in the pathophysiological
324
process of the infection. Thus, these cells can allow the development of relevant ex vivo co-
325
culture models, for example with dendritic cells or lymphocytes, as they express CCL20 in
326
response to H. pylori infection.
327
CXCL8 expression during H. pylori infection has long been described as being directly
328
linked to CagA translocation into epithelial cells (24, 25). Actually, CagA is not essential for
329
induction of this chemokine since CagA-deficient strains of H. pylori can likewise induce CXCL8
330
production by several cell lines at levels similar to those of wild-type strains (1, 26). In fact, it is
331
the presence of a functional TIVSS that plays a major role in the induction of CXCL8 and other
332
proinflammatory cytokines during H. pylori infection through NF-țB or AP-1 activation (27).
333
Several cag-PAI-dependent but cagA-independent NF-țB activation mechanisms have recently 15
334
been suggested, involving at least six different signaling pathways (27). One of them is the
335
internal recognition of translocated PG through TIVSS by the Nod1 protein, which activates NF-
336
țB signaling and thereby induces CXCL8 production (28). An alternative explanation for
337
chemokine production by gastric epithelial cells stimulated by B128ǻcagM strain is that since
338
TIVSS promotes intimate interactions between the bacteria and epithelial cells, endocytosis of
339
some H. pylori products, such as PG, is facilitated. This hypothesis was established by Viala et
340
al. to explain detection of radioactively labeled PG inside AGS cells exposed to ǻcagM-H. pylori
341
mutants (28). Furthermore, it seems that H. pylori can ensure PG translocation inside host cells
342
through the outer membrane vesicles that are constantly released by the bacterium (29). Recently,
343
it was suggested that CagL, via interactions with host integrins, can trigger pro-inflammatory
344
responses independently of CagA translocation or NOD1 signaling (30). As a result, the T4SS
345
apparatus per se could elicit host proinflammatory responses independently of its substrates (30).
346
Finally, chemokine production during H. pylori infection could also be induced by other bacterial
347
virulence factors. For example, Beswick et al. showed that the interaction of urease with the
348
surface receptor CD74 can activate NF-țB, which induces CXCL8 production (31). Smith et al.
349
reported that TLR2 and TLR5 are required for H. pylori-induced NF-țB activation and
350
chemokine expression by epithelial cells (5). It has also been suggested that H. pylori-induced
351
expression of TLR2 and TLR5 can qualitatively shift cag-PAI-dependent to cag-PAI-
352
independent pro-inflammatory signaling pathways leading to CXCL8 production in HEK293
353
cells transfected with TLR2 and TLR5 (32). The TLR2 ligand of H. pylori is controversial.
354
Indeed, it has also been suggested that lipopolysaccharide functions as a TLR2 ligand and
355
induces CXCL1-3 and CCL20 expression in MKN45 cells (33). Yet another study suggested that
356
H. pylori LPS does not activate TLR2 even at high concentrations of LPS (34). On the other 16
357
hand, H. pylori-heat shock protein 60 has been reported to induce CXCL8 via a TLR2 pathway in
358
monocytes (35). In addition, EGFR seems to play an important role in inflammatory reaction of
359
PGEC; this pathway can be activated by H. pylori independently of the presence of a functional
360
cag-PAI, as previously reported (36). Our data suggest that gastric epithelial cells can secrete
361
chemokines via cag-PAI-independent pathways, which have been shown to play a major role in
362
H. pylori-induced delayed inflammatory response.
363
Furthermore, expression of antimicrobial peptides in response to H. pylori has been
364
investigated as part of innate response and defense upon infection. Early cag-PAI-dependent BD2
365
mRNA expression was observed in PGEC infected with H. pylori whereas no significant
366
induction of this peptide was observed in AGS cells. In agreement, BD2 mRNA expression was
367
higher in the gastric mucosa of patients infected with cagA-positive strains than in those infected
368
with cagA-negative strains, a finding suggesting cag-PAI-dependent induction of this peptide.
369
BD2 was shown to be induced in the gastric epithelial tissues during H. pylori infection (37).
370
Furthermore, cag-PAI-dependent induction of BD2 was also observed in MKN45 cells infected
371
with H. pylori (38). The binding of BD2 on the surface of H. pylori cells was recently shown
372
(39), and it is a phenomenon that may explain the absence of detection of BD2 proteins in the cell
373
culture supernatants. Our results support the view that BD2 is expressed in response to early and
374
chronic H. pylori infection and that cag-PAI is involved in this antimicrobial peptide mRNA
375
induction.
376
Taken together, this new culture model of primary human gastric epithelial cells using
377
pieces of stomach enabled development of an H. pylori infection model and characterization of
378
the inflammatory profile generated upon infection. The cellular response of PGEC was
379
reproducible despite the different genetic backgrounds of the patients included in the study. 17
380
Furthermore, the inflammatory response of PGEC upon infection with H. pylori is closer to the in
381
vivo response characteristic of infected gastric mucosa than was that of previously described cell
382
lines, of which the response could be altered due to their tumoral physiology. The model also
383
supported the hypothesis of involvement of H. pylori TIVSS in eliciting an inflammatory
384
response at early phases of infection. At a later phase, our data supported the view that
385
inflammatory mediator expression and production are largely due to cag-PAI substrate
386
independent virulence factors. This largely novel human gastric epithelial cell model could be
387
used to investigate the interactions of stomach epithelium with inflammatory cells and to study
388
other functional aspects of infection with H. pylori in order to better characterize the pathological
389
processes associated with gastric infections.
390 391
Acknowledgements
392
This study was supported in part by a grant from the French Ministry of Health (PHRC
393
Pylorikine). Work in Ivo G Boneca laboratory was supported by ERC starting Grant
394
(PGNfromSHAPEtoVIR n°202283). We thank Emilie Peyrot and Damien Chassaing for
395
technical assistance and Jeffrey Arsham for English revision of the paper. There are no conflicts
396
of interest to disclose.
397 398
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399
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Figure legends
529 530
Figure 1. Immunodetection of CK18, MUC1, MUC5AC and TFF1 in PGEC cells and in AGS
531
cells. Scale bar: 50 μm.
532 533
Figure 2. Gene expression of PEGC and AGS cells stimulated by H. pylori. Cells were infected
534
by H. pylori B128 or B128ǻcagM at a MOI of 100 for 3h. QRT-PCR analysis was carried out on 24
535
total RNA of independent cultures from 5 different patients. mRNA expression levels are
536
expressed as the fold increase above unstimulated cultures. Data are represented as mean + SEM.
537
Statistically significant differences in inflammatory mediator production were studied between
538
infected cells and uninfected cells as well as between cells stimulated with H. pylori B128 and
539
B128ǻcagM. *p< 0.05 and **p
